Anti-HIV treatments have been very successful in suppressing viral load in HIV-infected patients. However, new therapeutic strategies are needed to circumvent the inevitable selection of viruses that are resistant to all currently used drugs. Of the 32 drugs approved for the treatment of HIV infection, only two block viral entry, and there are no approved drugs that target the HIV surface glycoprotein gp120. KD-247 is a promising humanized monoclonal antibody (mAb) that has been developed by collaborator Dr. Shuzo Matsushita and colleagues. It binds the V3 loop of gp120 and neutralizes extremely potently a broad panel of diverse clade B HIV-1 isolates. Moreover, KD-247 suppresses replication of viral strains that are resistant to all currently approved drugs. Based on its promising properties, KD-247 has entered Phase Ib clinical trials for the treatment of HIV-1 infections. However, KD-247 like other anti-V3 antibodies lacks cross- clade neutralization ability. The structural basis for the clade specificity of KD-247 is unclear. This proposal will focus on the use of structure-based design to construct second generation single-chain variable fragments (scFvs) of KD-247 with broader clade specificity. In preliminary results, we have solved the crystal structure of the unliganded antigen-binding fragment (Fab) of KD-247 at the highest resolution (1.55 E) reported for any humanized antibody to date. We have also obtained a cloned scFv based on wild-type KD-247 to be used for the construction of mutants and performed molecular modeling studies that will initially guide the selection of scFv mutations. We propose to extend these studies to pursue the following specific aims: 1. To determine why KD-247 neutralizes efficiently a broad spectrum of clade B isolates 2. To design, construct, and evaluate scFv KD-247 that effectively bind clade B AND non-clade B isolates Achieving the aims of this proposal will provide structural, biochemical, and virological knowledge that will direct the design and engineering of second generation anti-V3 antibodies with improved potency and broader neutralization ability. In addition, our expected high-resolution structures will also provide essential information about molecular protein-protein contacts important for antibody recognition of specific antigens. Structure-based anti-HIV mAb engineering is an innovative approach that can serve as a paradigm for the design of additional therapeutics. PUBLIC HEALTH RELEVANCE: This project will determine crystal structures of KD-247, a monoclonal antibody currently in clinical trials, that will help us understand why it works well with many, but not all types of HIV. This knowledge will help us design second generation antibodies with broader neutralization ability.